Mesoporous nanoperforators as membranolytic agents via nano- and molecular-scale multi-patterning

Plasma membrane lysis is an effective anticancer strategy, which mostly relying on soluble molecular membranolytic agents. However, nanomaterial-based membranolytic agents has been largely unexplored. Herein, we introduce a mesoporous membranolytic nanoperforators (MLNPs) via a nano- and molecular-scale multi-patterning strategy, featuring a spiky surface topography (nanoscale patterning) and molecular-level periodicity in the spikes with a benzene-bridged organosilica composition (molecular-scale patterning), which cooperatively endow an intrinsic membranolytic activity. Computational modelling reveals a nanospike-mediated multivalent perforation behaviour, i.e., multiple spikes induce nonlinearly enlarged membrane pores compared to a single spike, and that benzene groups aligned parallelly to a phospholipid molecule show considerably higher binding energy than other alignments, underpinning the importance of molecular ordering in phospholipid extraction for membranolysis. Finally, the antitumour activity of MLNPs is demonstrated in female Balb/c mouse models. This work demonstrates assembly of organosilica based bioactive nanostructures, enabling new understandings on nano-/molecular patterns co-governed nano-bio interaction.

The nanotube is composed of benzene bridged organosilica with a molecular scale ordering (schematically illustrated as below).In this case, only two kinds of beads are necessary to map the molecule: one for the [(CH)2] in benzene groups (Small Molecules, Adv.Theory and Sims. 2022, 5, 2100391) and one for the [O1/2-Si(C)-O1/2] groups (Soft Matter, 2022, 18, 7887-7896).According to the standard mode (Nature, 2002, 416, 304-307.),there are 60 -Si-and 30 benzene at each lap.After coarse graining, there are 60 [O1/2-Si(C)-O1/2] groups and 60 benzene [(CH)2] groups at each lap.The diameter and the length were set to 4.0 nm and 8.7 nm, respectively.The water particle has no charge and interacts with other particles through Lennard-Jones interactions.To simplify the simulation, we set the nanotube as a rigid structure.The quantum calculations were performed using restricted wB97XD with the 6-311+G* basis set.System preparation and general course-grained molecular dynamics simulation.
(1) Equilibrium conformation of the system.The initial structures and topologies for nanotubes were prepared using the Moltemplate program.The dimensions of the system are 34 nm × 34 nm × 35 nm.The lower membrane is 10 nm away from the bottom of the box, while the upper membrane is 20 nm away from the top.The systems were consisted of 4608 DPPC lipids and 200900 water molecules.The length and diameter of the nanotubes are 8.7 nm and 4.0 nm, respectively.Typically, three types of rigid nanotube, namely, single, sector and cone forms, were placed 12 nm above the upper membrane, separately.To keep consistent with experiment, the diameter of nanotube was set to 4 nm.In all simulations, the temperature was kept constant at 310 K by the Nosé-Hoover algorithm.The system was relaxed using an NPT (the constanttemperature, constant-pressure) ensemble for 10 ns.Short-ranged electrostatic interactions are cut off at 0.9 nm, while van de Waals interactions are cut off at 1.2 nm.All simulations were performed by the LAMMPS package.
(2) Non-equilibrium molecular dynamics (NEMD) simulation of nanotube(s) through DPPC membranes.After obtaining the stable system, the NVT (the constanttemperature, constant-volume) ensemble was used in simulation.We conducted the NEMD simulations with an external force (0.01 kcal mol -1 Å -1 ) since this allows gaining the phenomenon of membrane permeation within the limited simulation time.The timestep was set to 1 fs and the system was simulated for 1 ns.The pore areas as a function of distance between nanotube and bilayer membrane was calculated by python code.The radical distribution function was calculated by Visual Machine Design (VMD) software.

Molecular structure preparation and quantum chemistry calculation.
(1) Structure optimization.The molecular structure of DPPC lipid unit cell was built by Gauss View software.The molecules were optimized using the Gaussian 16 program with the wB97XD/6-311+G*.To simplify the system, all atoms but benzenes were removed in nanotube unit.Two benzenes were placed beside the DPPC lipid at a series of distance, namely, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 6.5 and 7.0 Å, respectively.(2) Binding energy calculation.After obtaining the optimized molecular structure, single point energy of each molecule as well as the combined structure was calculated using ωB97XD/6-311+G* with the Gaussian16 program.The binding energy equals to the difference between the single point energy of combined structure and that of single molecule.Considering the lack of perfect structure of nanotube, we did not optimize the complex.In simplest terms, the benzene dimer is part of the nanotube, we ignore the Si and O connected with benzene, therefore, the possible contact structure for benzene dimer is side contact, while the optimized structure is top contact.To make the calculation more reliable, we optimize the benzene and DPPC molecular with the ωB97XD/6-311+G* basis set and calculate the binding energy of three type of benzene groups in three typical positions as shown in Supplementary Figure 21.To make the calculation more reliable, we calculate the binding energy of three type of benzene groups in three typical positions.The binding energy of parallel structure is lowest at all three positions.And the highest binding energy value (-6.5 kcal/mol) of parallel structure in three position is lower than the lowest binding energy value (-4.5 kcal/mol) of perpendicular and parallel-perpendicular structure.Such multiple position sampling calculation can help to guarantee the interesting result that keeping the planes of each benzene ring positioned parallelly to the axial direction of the phospholipid molecule could lead to minimum binding energy.Remarkably, ωB97XD/6-311+G* is a middle lever basis set and the error is about 0.4 kcal/mol (ref: Computational andTheoretical Chemistry, 2016, 1098: 1-12).
Statistics and reproducibility.All statistical analyses were performed using GraphPad Prism 8.0.2.Data were analysed using unpaired two-tailed Student's t-tests for the calculation of P values.The number of replicates performed is indicated in each figure legend, where applicable.
Schematic illustration of the coarse graining model of nanotube in Martini force field, where purple ball present [O1/2-Si(C)-O1/2] group and grey ball present [CH]2 group.